Simulated space radiation-induced mutants in the mouse kidney display widespread genomic change.

Exposure to a small number of high-energy heavy charged particles (HZE ions), as found in the deep space environment, could significantly affect astronaut health following prolonged periods of space travel if these ions induce mutations and related cancers. In this study, we used an in vivo mutagenesis assay to define the mutagenic effects of accelerated 56Fe ions (1 GeV/amu, 151 keV/μm) in the mouse kidney epithelium exposed to doses ranging from 0.25 to 2.0 Gy. These doses represent fluences ranging from 1 to 8 particle traversals per cell nucleus. The Aprt locus, located on chromosome 8, was used to select induced and spontaneous mutants. To fully define the mutagenic effects, we used multiple endpoints including mutant frequencies, mutation spectrum for chromosome 8, translocations involving chromosome 8, and mutations affecting non-selected chromosomes. The results demonstrate mutagenic effects that often affect multiple chromosomes for all Fe ion doses tested. For comparison with the most abundant sparsely ionizing particle found in space, we also examined the mutagenic effects of high-energy protons (1 GeV, 0.24 keV/μm) at 0.5 and 1.0 Gy. Similar doses of protons were not as mutagenic as Fe ions for many assays, though genomic effects were detected in Aprt mutants at these doses. Considered as a whole, the data demonstrate that Fe ions are highly mutagenic at the low doses and fluences of relevance to human spaceflight, and that cells with considerable genomic mutations are readily induced by these exposures and persist in the kidney epithelium. The level of genomic change produced by low fluence exposure to heavy ions is reminiscent of the extensive rearrangements seen in tumor genomes suggesting a potential initiation step in radiation carcinogenesis

Simulated space radiation-induced mutants in the mouse kidney display widespread genomic change.

Exposure to a small number of high-energy heavy charged particles (HZE ions), as found in the deep space environment, could significantly affect astronaut health following prolonged periods of space travel if these ions induce mutations and related cancers. In this study, we used an in vivo mutagenesis assay to define the mutagenic effects of accelerated 56Fe ions (1 GeV/amu, 151 keV/μm) in the mouse kidney epithelium exposed to doses ranging from 0.25 to 2.0 Gy. These doses represent fluences ranging from 1 to 8 particle traversals per cell nucleus. The Aprt locus, located on chromosome 8, was used to select induced and spontaneous mutants. To fully define the mutagenic effects, we used multiple endpoints including mutant frequencies, mutation spectrum for chromosome 8, translocations involving chromosome 8, and mutations affecting non-selected chromosomes. The results demonstrate mutagenic effects that often affect multiple chromosomes for all Fe ion doses tested. For comparison with the most abundant sparsely ionizing particle found in space, we also examined the mutagenic effects of high-energy protons (1 GeV, 0.24 keV/μm) at 0.5 and 1.0 Gy. Similar doses of protons were not as mutagenic as Fe ions for many assays, though genomic effects were detected in Aprt mutants at these doses. Considered as a whole, the data demonstrate that Fe ions are highly mutagenic at the low doses and fluences of relevance to human spaceflight, and that cells with considerable genomic mutations are readily induced by these exposures and persist in the kidney epithelium. The level of genomic change produced by low fluence exposure to heavy ions is reminiscent of the extensive rearrangements seen in tumor genomes suggesting a potential initiation step in radiation carcinogenesis

Cytotoxicity for cloning efficiencies for kidney epithelial cells removed from charged particle-exposed kidneys.

<p>Cloning efficiencies (CE) were determined for kidneys exposed to Fe ions and protons and plotted relative to cloning efficiencies for untreated kidneys. Data for 3–5 Gy protons were taken from a prior study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180412#pone.0180412.ref013" target="_blank">13</a>].</p

Loss of heterozygosity (LOH) patterns reveal different mutational events.

<p><b>A.</b> The relative locations of polymorphic loci on mouse chromosome 8 that differ between the C57BL/6 and DBA2 mouse strains and were used to deduce the specific mutation present in each <i>Aprt</i> mutant examined in this study. The B6 (C57BL/6) derived chromosome contains a knockout (KO) <i>Aprt</i> allele that is non-functional. The D2 (DBA/2) derived chromosome contains an expressed, wild type <i>Aprt</i> allele, which is the target of DAP selection (i.e. loss of expression of this <i>Aprt</i> allele due to a mutational event allows a cell to grow in the presence of DAP in the culture medium). The numbers on the left side identify the chromosome 8 microsatellite loci that were examined in this study. These numbers omit the D8Mit prefix. <b>B.</b> The PCR-based molecular analysis for loss (closed circles) or retention (open circles) of heterozygosity for polymorphic microsatellite sequences on mouse chromosome 8 in the <i>Aprt</i> mutant cells yields LOH patterns that can be used to classify each mutational event into one of 8 different categories. These are intragenic events (IE), apparent mitotic recombination (MR), interstitial deletion of <i>Aprt</i> polymorphic locus only (DEL 1), multilocus deletion (DEL > 1), chromosome loss (CL), and discontinuous LOH (DLOH). See text for more details.</p

Translocations involving chromosome 8 in Fe ion-induced mutants with the mitotic recombination LOH pattern^a.

<p>Translocations involving chromosome 8 in Fe ion-induced mutants with the mitotic recombination LOH pattern<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180412#t003fn001" target="_blank">^a</a>.</p

Mutant frequencies and cytotoxicity in charged particle exposed kidney epithelial cells.

<p>Mutant frequencies and cytotoxicity in charged particle exposed kidney epithelial cells.</p

Analysis of genomic LOH in mutants from ⁵⁶Fe ion and proton irradiated kidneys^a.

<p>Analysis of genomic LOH in mutants from ⁵⁶Fe ion and proton irradiated kidneys<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180412#t004fn001" target="_blank">^a</a>.</p

Genomic SNP analysis for <i>Aprt</i> mutants from Fe ion exposed kidneys.

<p>DNA from each mutant shown was analyzed for loss or retention of heterozygosity for multiple SNPs along each chromosome and the molecular patterns used to identify LOH tracts on affected chromosomes and used to determine the type of mutation. A color key is provided at the bottom left of the figure. The <i>Aprt</i> mutants were selected for different types of mutations affecting chromosome 8 and a subset were identified with FISH analysis for translocations (marked with black). Mutant 932RK1 has an intragenic mutation affecting <i>Aprt</i> expression. This figure also shows results for two spontaneous <i>Aprt</i> mutants (0 Gy) and two non-selected clones from kidneys exposed to 0.5 Gy. Chromosome 4 is omitted from this figure (see text).</p

Estimated frequencies for the different mutational events.

<p>Relative mutation frequencies for different mutational events, with the overall spontaneous mutant frequency set at 1 X 10⁻⁴, at the different Fe ion doses were multiplied by the relative percentages of each mutational event, as defined by the LOH analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180412#pone.0180412.t002" target="_blank">Table 2</a>), to estimate the mutation frequencies for each event. Intragenic events (IE), chromosome loss (CL), mitotic recombination (MR), interstitial deletion of <i>Aprt</i> polymorphic locus only (DEL), multilocus deletion (DEL > 1), and discontinuous LOH (DLOH). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180412#pone.0180412.g003" target="_blank">Fig 3B</a> for representative LOH patterns for each category.</p

Distribution of LOH events on non-selected chromosomes in the <i>Aprt</i> mutants from Fe ion and proton-exposed kidneys.

<p>A Chi-square analysis was used to assess the distribution of non-selected chromosomes LOH events in <i>Aprt</i> mutants from Fe ion and proton-exposed kidneys. A value of 8.06 or more (dotted line) is required to conclude a chromosome has been differentially affected based on a Bonferroni adjustment. Only chromosome 14 met this threshold.</p